Evaluation of Anti Urolithiatic Activity of Dolichos biflorus Seed Extract by Using Ethylene Glycol Induced Model

In the phytochemical screening, it was found that Dolichos Biflorus seed extract contained the presence of tannins steroids, protein, flavonoids, terpenoids, mucilage, saponin, and carbohydrate, but no alkaloids fixed oil. So, this plant has highly diuretic activity. Which can use for the treatment of urolithiasis. Objective: The Present study aims to investigate the anti-Urolithiatic Urolithiatic markers such as calcium, creatinine, phosphorus, uric acid, alkaline phosphatase, BUN (Blood Urea Nitrogen), and potassium were normal in animals receiving plant extract and prevent renal cell injury. Conclusion: All its findings and phytoconstituents existing inside the extract must stay the viable chemical materials involved in the prevention of Urolithiatic.


INTRODUCTION
The stones in the kidneys are characterised by solid particles formed in the kidneys and urinary system. However, in some cases, these stones can be minute and go through the blood circulation and pass out the body through the urinary tract with urine. In this case, the formation of kidney stones has no harm or damage to the body organs or tissues. In the contrast, if the stones are bigger, they will block the urine flow and cause pain that required medical treatment. Urolithiasis is one of the most common diseases of the human urinary tract [1] and is associated with calculus or stone formation in the urinary system but kidney calculus often arises in the kidney. Kidney stones form when urine contains a more crystal forming substance such as calcium, oxalate, and uric acid. These crystal forming agents come together to form crystals [2].
Formation of renal crystal is a multifactorial process that may relate to diet, urinary tract infection, altered urinary solutes and colloids, decrease urinary drainage and urinary stasis, and prolonged immobilization of Randall's plaque and microlith [3].
The metabolism of ethylene glycol metabolized by the human body results in toxic compounds such as glycolaldehyde, glycolate and glyoxylate which result in destroying of tissue due to the deposition of the calcium oxalate deposition. Specifically, a high anion gap causes metabolic acidosis, lactic acidosis and hypocalcaemia. Oxalate acid is associated with calcium to form calcium oxalate crystals, which deposit in the kidney causing haematuria and the presence of the protein urea, increasing creatinine and eventually renal failure [4].
Decoction of seeds is used in diarrhoea, and haemorrhage from bowels, and is given to females during parturition to promote the discharge of the lochia. Pulse is a demulcent in calculus affection and coughs. A soup is a diet in sub-acute cases of enlarged liver and spleen. As a home remedy, kulthi has been used in dysuria, bleeding piles, vaginal bleeding, and leucorrhoea. Its use in reducing obesity is also recognized [5]. In short, there is a good level of traditional and experimental evidence to support various claims and advantages of this widely available plant [6]. Several medications prescribed for preventing the recurrence include thiazide diuretics and alkali citrate. However, the effectiveness of these drugs is still not clinically proven [7].
Using herbal medicine and plant extracts has been widely known as an alternative to chemical or synthetic drugs with a lesser side effect. This leads to the fact that using herbal medicine techniques is corresponding with the return to natural remedies with less or absent side effects. The urinary stones disease is known throughout history and is very common in the Northern part of India [8]. It is also used to reduce crystalluria and to lyse stones. The powdered seeds are used as a poultice to induce sweating [10].

Plant Information
Dolichos biflorus is commonly used in herbal and traditional medicine with remarkable healing properties. Seeds of this plant are widely used treatment of several ailments besides being used as a tonic, astringent, and diuretic [11].

Plant Collection and Extract Preparation
The seeds of the plant were obtained from the authorized commercial dealer (Shree Mahadev enterprises, 35, RadhikaVihar, Krishna Nagar, Mathura-281004).

Preparation of extract
The seeds of the plant Dolichos biflorus were collected and washed using fresh water and dried under shade. The seeds are crushed to a fine powder after drying. The chemical compounds present in the seeds were extracted with methanol by soxhlation. The solvent is then evaporated by using a rotary evaporator and the phytochemicals were collected and stored for further analysis.

Experimental Animals
Wistar rats Male (8-10 weeks old) weighing 150-200g were included in this study. Before starting the experiment, the rats were maintained in the laboratory for adjustment and adaptation for 7 days. The experimental rats were maintained in ventilated cages at a photoperiod of 12:12 and a temperature of 25 ± 2°C. The animals had easy access to ad libitum standard chewable food and water.

Ethylene glycol-induced urolithiasis
Groupings were done in the following manner, Where N = 6 animals in each group (Chart 1).
At the end of the treatment, serum samples were collected and the animals were sacrificed by using a high dose of pentobarbitone sodium for histopathology and antioxidant analysis of the kidney. Blood samples were withdrawn by cardiac puncture and retroorbital routes and samples were centrifuged at 2500 rpm for 15 minutes and examined. The parameters;

Ethylene glycol induced
Uurolithiasis in Rat's Model The euthanized rats were conducted through a high dose of Pentobarbital followed by scarification. Kidneys of each animal were dissected and chopped into small parts followed by preservation and fixation in 10% formalin for 48 hours. Alcohol was used for tissue dehydration, embedded in paraffin, cut in a microtome and stained with Haematoxylin-Eosin dye. The mounted slides were examined under the microscope and compared to the slides from the control group. Determination of biochemistry markers was carried out following the standard protocols and procedures in the manufacturer's instructions.

Statistical Analysis
The data of the present study was analysed descriptively for the mean and standard error mean (Mean ± S.E.M.) of the 6 replicates in each experimental group. All statistical analyses were carried out using the Graph Pad Prism software package (version 5). The one-way analysis of variance (ANOVA) was utilized to examine the significant differences in the means at the significance level of P<0.05. The multiple comparison test of the Tukey HSD test was used for pairwise comparison.

RESULTS
The Control group received an equivalent volume of "vehicle" only, the Disease control group received "Ethylene glycol" (0.75%v/v) in water, the Standard group received "Cystone" (750mg/kg,p.o.) and the Test drug, "dolichos biflorus" extract received low dose 150mg/kg & high dose 300 mg/kg p.o. respectively.                  The histological profile of the kidney tissue proved the antiurolithiatic effect. Interestingly, the sections of the rat's kidneys in the control group showed the formation of crystals. In addition to that, epithelial cells of the kidney showed remarkable tubular dilatation and damage as illustrated by the gaps in the tissue. However, in the treated group, the rat's kidney exhibited less crystal formation. Furthermore, less necrosis and tubule dilatation were scarce. The damage caused by the formation of the kidney stones is ultimately affecting the renal function in the control group as proven by the markers of glomerular and tubular damage: elevated levels of BUN, uric acid, urea, and serum creatinine were lower in the treated animals. Inflammation and tissue damage are because exposing them to phosphate and calcium phosphate crystals results in the formation of reactive oxygen species, development of oxidative stress, lipid peroxidation, and depletion of antioxidant enzymes. In the same context, the damage to the epithelial tissue of the kidney enhances crystal retention and eventually promotes the formation of kidney stones on the epithelial tissue surface. One important explanation of the plant extract mechanism is that antioxidant compounds can enhance the antioxidant enzymes in the kidney and constrain cell injury or damage.

Estimation of Alanine Amino Transferases
The herbal or traditional medicines are used through oral intake similar to the procedure of anti urolithiatic activity evaluation of L. procumbens against ethylene glycol-induced renal calculi in rats. The justification for this study using rat males is the urinary system of the males is quite similar to humans in inducing and formation of urolithiasis. In addition, several studies reported lesser kidney stone formation in rat females compared to males [15].

CONCLUSION
This study describes the anti-urolithiatic activity of Dolichos biflorus methanolic seeds extract in Ethylene glycol induced urolithiatic in a rat's model.
From the present study, we conclude the preliminary phytochemical analysis of Dolichos Biflorus Lin. indicated the presence of Alkaloids, Flavonoids, Proteins, Saponins, Terpenoids, Phytosterols, Carbohydrates and Fatty acids and it has a potent diuretic effect. BUN, uric acid, urea and serum, and creatinine that was lowered in animals receiving plant extract. The protection of the Kidney with this treatment is ideal for urolithiasis patients.
All other urolithiasis markers like calcium, BUN, potassium, and creatinine showed a dosedependent manner concerning their control group. The alkaline phosphatase, calcium, and uric acid also improved significantly.
Histology of kidneys in urolithiatic animals showed that necrosis, as well as tubule dilatation, was very limited.
The high dose of Dolichos biflorus (300mg/kg, PO) is showing a better effect as compared to the low dose of (150mg/kg, PO) and the standard drug (750 mg/kg, PO).

CONSENT
It is not applicable.

ETHICAL APPROVAL
This study had been ethically approved by the (Institutional Animal Ethical Committee) of Karnataka College of Pharmacy, Bangalore. Which conducted the meeting for M.pharm Students on the date (18/12/2021) before taking part in the study, the committee approved for use of the animals in the experiment and provided the registration number (Reg. Number: IAEC/ 09/21-22/10/18/12/21) to start the experiment on animals.